Project Summary/Abstract: Mislocalization and aggregation of RNA binding proteins (RBPs) is observed in many neurodegenerative diseases (ND) including Frontotemporal Dementia (FTD), Amyotrophic Lateral Sclerosis (ALS) and Alzheimer?s Disease (AD). A notable example is the mislocalization and aggregation of TDP-43. Despite growing evidence, including rare mutations in the protein itself, pointing to the central role of TDP-43 aggregation in the disease etiology of ALS and FTD, the genetic networks that control this event are poorly understood. Many previous screens for identifying modifiers of the effect of mutant proteins associated with neurodegenerative disease (NDs) use toxicity as the phenotypic readout, yet it is unclear how the acute cellular toxicity used in such screens relate to the chronic long-term toxicity observed in NDs. Our lab has developed a cellular reporter that enables accurate quantification of TDP-43 aggregation status at the single cell level by flow cytometry using a method called Pulse Shape Analysis. We propose to use this reporter to conduct pooled, marker-based, genome-wide CRISPR screens to map the genetic network that directly control TDP43 aggregation in human cell lines, neuronal progenitor cells and neurons. In contrast to arrayed, imaging-based screens, our approach is highly scalable, and will serve as a blueprint to study the aggregation of additional RNA binding proteins in both a physiological (e.g. formation of stress granules) and disease context. We plan to use this pilot study to establish a research program that will aim to systematically map the control network of physiological and pathological aggregation of RNA binding proteins and explore the prospect of modulating this network as a therapeutic approach.